The protein serine-threonine phosphatase 2A (PP2A) family of enzymes participates in a wide spectrum of cellular functions including cell growth, differentiation, cell cycle progression, and apoptosis. Multiple regulatory mechanisms control the activity of PP2A in vivo. Preliminary findings from the applicant's laboratory have revealed a novel post-translational modification of the PP2A catalytic subunit (PP2Ac), namely monoubiquitination, which has recently been shown to play an essential role in directing the subcellular localization of target proteins. Consistent with these findings, a recent study from the applicant's collaborator has revealed defective ubiquitin-dependent regulation of PP2Ac as the underlying cause of Opitz Syndrome (OS), a disorder characterized by malformations of the ventral midline (Nat. Gen. 29:287, 2001). Mutations in the Midi gene, which are found in OS patients, lead to marked accumulation of microtubule-associated PP2Ac. This defect likely represents an impairment of the E3 ubiquitin ligase activity of the Midi protein that normally facilitates PP2Ac polyubiquitination and subsequent degradation. The specificity of Midi for PP2Ac is defined by its interaction with alpha4, a known interacting partner of PP2Ac that also has been linked to another OS-like condition known as FG syndrome. The applicant hypothesizes that Mid1/alpha4-dependent mono- and polyubiquitination of PP2Ac determine the enzyme's subcellular localization and degradation, respectively, thereby influencing phosphatase activity towards key signaling enzymes involved in the control of cell growth and proliferation including p70 S6 kinase and mitogen-activated protein kinases. To address this central hypothesis, the applicant proposes a series of complementary biochemical, immunological, and genetic investigations to elucidate the molecular mechanism, regulation, and function of PP2Ac ubiquitination. These studies will provide fundamental mechanistic insights into the pathobiochemistry of OS and FG syndrome, as well as the role of this posttranslational modification in the control of signal transduction pathways mediating cell growth and survival. Given the importance of PP2A in the maintenance of cell homeostasis, the proposed studies will undoubtedly identify novel targets to manipulate phosphatase activity within specific signaling pathways, and potential therapeutics for diseases affecting normal cell growth and survival.